Osteocalcin, one of the very few osteoblast-specific proteins, has several features of a hormone. For instance, it is synthesized as a pre-pro-molecule and is secreted in the general circulation (Hauschka et al., 1989, Physiol. Review 69:990-1047; Price, 1989, Connect. Tissue Res. 21:51-57 (discussion 57-60)). Because of their exquisite cell-specific expression, the osteocalcin genes have been intensively studied to identify osteoblast-specific transcription factors and to define the molecular bases of bone physiology (Ducy et al., 2000, Science 289:1501-1504; Harada & Rodan, 2003, Nature 423:349-355).
Osteocalcin is the most abundant non-collagenous protein found associated with the mineralized bone matrix and it is currently being used as a biological marker for clinical assessment of bone turnover. Osteocalcin is a small (46-50 amino acid residues) bone specific protein that contains 3 gamma-carboxylated glutamic acid residues in its primary structure. The name osteocalcin (osteo, Greek for bone; calc, Latin for lime salts; in, protein) derives from the protein's ability to bind Ca2+ and its abundance in bone. Osteocalcin undergoes a peculiar posttranslational modification whereby glutamic acid residues are carboxylated to form gamma-carboxyglutamic acid (Gla) residues; hence osteocalcin's other name, bone Gla protein (Hauschka et al., 1989, Physiol. Review 69:990-1047).
Mature human osteocalcin contains 49 amino acids with a predicted molecular mass of 5,800 kDa (Poser et al., 1980, J. Biol. Chem. 255:8685-8691). Osteocalcin is synthesized primarily by osteoblasts and ondontoblasts and comprises 15 to 20% of the non-collagenous protein of bone. Poser et al., 1980, J. Biol. Chem. 255:8685-8691 showed that mature osteocalcin contains three carboxyglutamic acid residues which are formed by posttranslational vitamin K-dependent modification of glutamic acid residues. The carboxylated Gla residues are at positions 17, 21 and 24 of mature human osteocalcin. Some human osteocalcin has been shown to contain only 2 Gla residues (Poser & Price, 1979, J. Biol. Chem. 254:431-436).
Osteocalcin has several features of a hormone. Ducy et al., 1996, Nature 382:448-452 demonstrated that mineralized bone from aging osteocalcin-deficient mice was two times thicker than that of wild-t e. It was shown that the absence of osteocalcin led to an increase in bone formation without impairing bone resorption and did not affect mineralization. Multiple immunoreactive forms of human osteocalcin have been discovered in circulation (Garnero et al., 1994, J. Bone Miner. Res. 9:255-264) and also in urine (Taylor et al., 1990, J. Clin. Endocrin. Metab. 70:467-472). Fragments of human osteocalcin can be produced either during osteoclastic degradation of bone matrix or as the result of the catabolic breakdown of the circulating protein after synthesis by osteoblasts.
The identification in recent years of novel organs influencing bone physiology expanded the spectrum of questions studied in skeletal biology. An example of this is the regulation of bone mass accrual by the brain that was first revealed by studying the mechanisms whereby the adipocyte-derived hormone leptin decreases bone mass accrual in all species tested (Ducy et al., 2000, Cell 100:197-207; Pogoda et al., 2006, J. Bone and Mineral Res. 21:1591-1599; Elefteriou et al., 2004, Proceedings of the National Academy of Sciences of the United States of America 101:3258-3263; Vaira et al., 2012, Neuroscience Biobehavioral Rev. 29:237-258). The use of cell-specific gene deletion models revealed widespread evidence that leptin signals in brainstem neurons to prevent synthesis of serotonin, a neurotransmitter that decreases the activity of the sympathetic nervous system, an inhibitor of bone mass accrual (Takeda et al., 2002, Cell 111:305-317; Yadav et al., 2009, Cell 138:976-989; Oury et al., 2010, Genes & Development 24:2330-2342, Genes Dev. 24:2330-2342). What underlines best the importance of this function of brain-derived serotonin is the fact that selective serotonin reuptake inhibitors (SSRIs) that increase the local concentrations of serotonin in the brain (Gardier et al., 1996, Fundamental Clin. Pharmacol. 10:16-27) have deleterious effects on bone mass in humans.
A second development of significance in skeletal biology has been the demonstration that bone is an endocrine organ secreting at least two hormones. One of them, osteocalcin, is made by the osteoblast, the bone forming cell, and promotes several functions apparently unrelated to bone health such as energy expenditure, insulin secretion, insulin sensitivity, and, in males, testosterone synthesis (Lee et al., 2007, Cell 130:456-469; Oury et al., 2011, Cell 144:796-809). The latter function occurs following the binding of osteocalcin to a specific receptor, gprc6a, on Leydig cells (Oury et al., 2011, Cell 144:796-809).
OST-PTP is the protein encoded by the Esp gene. The Esp gene was originally named for embryonic stem (ES) cell phosphatase and it has also been called the Ptprv gene in mice. (Lee et al, 1996, Mech. Dev. 59:153-164). Because of its bone and testicular localization, the gene product of Esp is often referred to as osteoblast testicular protein tyrosine phosphatase (OST-PTP). OST-PTP is a large, 1,711 amino acid-long protein that includes three distinct domains. OST-PTP has a 1,068 amino-acid long extracellular domain containing multiple fibronectin type III repeats.
Esp expression is restricted to ES cells, the gonads, and the skeleton. In the gonads, Esp is specifically expressed in Sertoli cells of the testis and coelomic epithelial cells of the ovaries. During development, Esp is initially expressed in the apical ectodermal ridge of the limbs. Later during embryonic development and after birth, its expression becomes restricted to pre-osteoblasts and osteoblasts (i.e., Runx2-positive cells) of the perichondrium and periosteum.
Protein tyrosine phosphatase-1B (PTP-1B) is an ˜50 kd intracellular protein present in abundant amounts in various human tissues (Charbonneau et al., 1989, Proc. Natl. Acad. Sci. USA 86:5252-5256; Goldstein, 1993, Receptor 3:1-15).
Gprc6a is a receptor that belongs to the C family of GPCRs (Wellendorph and Brauner-Osborne, 2004, Gene 335:37-46) and has been proposed to be a receptor for amino acids or for calcium in the presence of osteocalcin as a cofactor, and for androgens (Pi et al., 2008, PLoS One.3:e3858; Pi et al., 2005, J. Biol. Chem. 280:40201-40209; Pi et al., 2010, J. Biol. Chem. 285:39953-39964).
Embryonic development is affected by a variety of environmental signals. In particular, both clinical outcome studies and experimental evidence gathered in model organisms concur to indicate that the mother's health during pregnancy is an important determinant of embryonic development (Osorio et al., 2012, Nature Rev. Endocrinol. 8:624; Lawlor et al., 2012, Nature Rev. Endocrinol. 8:679-688; Challis et al., 2012, Nature Rev. Endocrinol. 8:629-630). By definition, any direct maternal influence on vertebrate embryonic development occurs through the placenta, an organ allowing the transfer of circulating molecules from the mother to the embryo. To date however, molecules either made in the placenta or by the mother, crossing the placenta and that would affect development of the brain of the pup, have not been identified. This is an important question considering that a growing number of epidemiological studies suggest that maternal health may also be a risk factor for neurologic and psychiatric diseases in the offspring (Wadhwa et al., 2001, Prog. Brain Res. 133:131-142; Van den Bergh et al., 2005, Neurosci. Biobehavioral Rev. 29:237-258; Weinstock, 2008, Neurosci. Biobehavioral Rev. 32:1073-1086).